title: Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases.
abstract: Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect's digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 μM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.
authors.affiliation: Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, CEP 91501-970 Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Marina S
authors.initials: MS
authors.lastname: Defferrari
authors.forename: Diogo R
authors.initials: DR
authors.lastname: Demartini
authors.forename: Thiago B
authors.initials: TB
authors.lastname: Marcelino
authors.forename: Paulo M
authors.initials: PM
authors.lastname: Pinto
authors.forename: Celia R
authors.initials: CR
authors.lastname: Carlini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D032449:Canavalia / Q000201:enzymology / Q000633:toxicity*; D056668:Cathepsin L / Q000737:chemistry / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003374:Coumarins / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004063:Digestion; D004151:Dipeptides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D020062:Heteroptera / Q000187:drug effects / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D009758:Nymph / Q000187:drug effects* / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D014510:Urease / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity
chemical_list: D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007306:Insecticides; D010446:Peptide Fragments; D010940:Plant Proteins; D011498:Protein Precursors; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D056668:Cathepsin L; D014510:Urease; D007930:Leucine; C024974:E 64